View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Springer - Publisher Connector Parasitol Res (2016) 115:681–685 DOI 10.1007/s00436-015-4787-2

ORIGINAL PAPER

Reclassification of pogonae Walden (2009) as Choleoeimeria pogonae comb. nov.(: )

Klaudiusz Oktawian Szczepaniak1 & Krzysztof Tomczuk1 & Anna Lojszczyk-Szczepaniak1 & Wojciech Lopuszynski1

Received: 20 September 2015 /Accepted: 6 October 2015 /Published online: 14 October 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com

Abstract The presented paper provides a reclassification of izing P. vitticeps have been described (McAllister et al. 1995; Eimeria pogonae from Pogona vitticeps into the correct genus Walden 2009). Choleoeimeria. A description of exogenous and endogenous amphiboluri is a parasite with relatively strict host stages of biliary coccidium is given. Sporulation of the oocysts specificity for the Australian agamid lizard from the genus was endogenous. The mature oocysts contained four sporo- Pogona, reported in both wild and captive populations. This cysts each with two sporozoites. Oocysts were ellipsoidal in species was first described by Cannon (1967), in Pogona shape, with average length/width ratio 1.7 and measured 28.4 barbata, and re-described by McAllister et al. (1995)in (SD1.5)×16.8 (SD 1.5). The micropyle, residuum, and polar P. vitticeps. Isosporosis of young bearded dragons is usually granules were absent from the sporulated oocysts. Ovoidal in associated with high mortalities while in adult individuals, shape, sporosysts without Steida bodies contained residuum I. amphiboluri seem to be a parasite of low pathogenicity. In and two elongated and boat-shaped sporozoites. The endoge- the whole life cycle of the endogenous stage, this parasite nous stages of the were located mainly in the epithe- appears to be limited to the epithelial cells of the intestine. lium of bile ducts; however, single-epithelium cells of the Novel Eimeria sp. oocysts in faecal samples of P. vitticeps gallbladder were also infected. obtained from two captive breeding populations were ob- served by Walden (2009). Approximately 80 % of these oo- Keywords Biliary coccidium . Bearded dragon . Liver . cysts were sporulated in fresh faeces. All oocysts were ellip- Coccidiosis soidal with an average size and shape index (n=20) of 27.2× 15.1 μm and 1.8, respectively. According to the morphologi- cal characteristics of the detected oocysts, the author placed this new species within the genus Eimeria and proposed the Introduction name Eimeria pogonae after the host’s generic name. Their description was only limited to the exogenous stages, but One of the most popular lizards kept individually as an exotic nothing was reported about the infection site and endogenous pet is the bearded dragon (Pogona vitticeps). This is a species development. However, for a correct taxonomic classification native to Central Australia, widely available in world pet of a coccidian, it is fundamental to follow its host trade. Common endoparasites detected in bearded dragons localization and mode of development in the endogenous are coccidia (Raiti 2012). To date, only two coccidia parasit- stages. Some reptilian coccidia which undergo endogenous devel- opment in the gallbladder epithelium, previously described as Eimeria, were reclassified by Paperna and Landsberg (1989) * Klaudiusz Oktawian Szczepaniak as a separate genus referred to as Choleoeimeria. The molec- [email protected] ular separation of the genera Eimeria and Choleoeimeria was confirmed by Jirků et al. (2002). Despite the fact that many 1 Faculty of Veterinary Medicine, University of Life Sciences in species of Choleoeimeria were recognized in different reptil- Lublin, Akademicka 13, Lublin 20-950, Lubelskie, Poland ian families to date (Abdel-Baki 2014; Abdel-Baki et al. 2014; 682 Parasitol Res (2016) 115:681–685

Al-Quraishy et al. 2013), there has been no study on deter- (24/24). All sporulated oocysts obtained from the intestinal mining the occurrence of Choleoeimeria spp. in bearded content and bile have the same morphotype and did not differ dragons. significantly in size, but the number of oocysts in the bile and The presented paper provides a morphological and intestinal content varied. Numerous oocysts were observed in morphometrical characterization of oocyst structures of the the gallbladder while only single ones in the intestine. A his- coccidians isolated from the gallbladder and intestinal content tological section of the gallbladder demonstrated that most of of P. vitticeps. Further, a description is given of localization of the oocysts from the bile were aggregated in dense clusters. endogenous development and histopathological changes in Oocysts isolated from the intestine were fully sporulated the epithelial cells of the gallbladder. According to the oocyst (four sporocysts with two sporozoites each), while oocysts morphology and infection site, we classified the coccidium as from the gallbladder were in different stages of development. belonging to the genus Choleoeimeria. Forty percent of the observed oocysts were developed to the stadium of four sporocysts per oocyst. In 35 % of the oocysts, sporozoites were released from the sporocysts and eight sep- Materials and methods arate sporozoites were seen inside an oocyst. Only 2 % of the oocysts were unsporulated. One percent of the oocysts, pres- A total of 24 dead adults, naturally infected with coccidian ent in bile smears from all the examined lizards, were rup- bearded dragons, were delivered to the laboratory of Depart- tured, while single, free sporozoites released from the oocysts ment of Parasitology and Invasive Diseases, Faculty of Veter- were observed in the bile. The remaining oocysts (22 %) were inary Medicine, University of Life Sciences, Lublin, Poland. empty or contained numerous granuloma structures, but the All of the animals came from a commercial farm of captive wall of these oocysts seemed to be unaltered. . Necropsy examinations and samples collections were The mature oocysts were ellipsoidal in shape, measured done within a few hours of the lizards’ death. Their intestinal 28.4 (SD 1.5)×16.8 (SD 1.5) (25.6–32.3×13.6–21.4). The contents or faeces (obtained from rectum or/and cloaca) were length/width ratio (L/W) was 1.7 (SD 0.1) (1.4–2.2). The wall examined using Sheather’s sugar flotation method. The gall- was double-layered measuring 0.7 (0.6–1.1) in thickness, with bladder contents were examined in wet mounts. Sporulated a smooth surface texture. The micropyle, residuum and polar oocysts were observed via a light microscope fitted with granules were absent from the sporulated oocysts. Ovoidal in Nomarski interference-contrast (DIC). For morphometric da- shape, sporocysts without Steida bodies contained residuum ta, only sporulated oocysts (at least 50 from each individual) and two sporozoites. The average sporocyst size was 12.5 (SD were used. Measurements were made from photographs ob- 1.5)×6.6 (SD 0.5) (9.4–14.7×5.3–7.4); the length/width ratio tained of the living oocysts, without any signs of degeneration, (L/W) was 1.8 (SD 0.2) (1.4–2.3). On the surface of sporocyst in accordance with guidelines set by Duszynski and Wilber wall, longitudinal suture was seen. Sporozoites were elongat- (1997). ed and boat-shaped, slightly tapering at the anterior and pos- To study the endogenous stages, samples of the intestines, terior ends, possessing a smooth surface, measured 13.3×3.5 liver and gallbladder were fixed in 10 % formalin, pH=7.2 for (12.7–14.1×3.2–3.7), with length/width ratio 3.8 (3.7–3.9). 24 h, and routinely processed and embedded in paraffin Each of them had one distinct posterior refractile body 2.8 blocks. The histological sections were stained with (2.5–3.0) (Fig. 2a–c). haematoxylin and eosin. All measurements were given in The results of the histopathological examination confirmed micrometres (μm). endogenous development of coccidia in the epithelium of the gallbladder and bile ducts. Numerous endogenous stages of coccidia were seen in the epithelial cells of the medium and Results large bile ducts, while only single host cells were infected in the gallbladder. A microscopic analysis revealed various en- Sixteen necropsied animals were in poor nutritional condition dogenous stages of coccidia (Figs. 3). The infected cells were with visible signs of emaciation and dehydration, while rest displaced into the lumen of bile duct or gallbladder and at- eight were cachexic. A necropsy revealed underdeveloped fat tached to the basement membrane through a stalk of cyto- bodies in all lizards. Slight jaundice in the mucous and serous plasm. In many regions of the gallbladder, mucosal abnormal- membranes was seen in 13 individuals. Twelve lizards had ities such as focal pseudostratification, epithelial hypertrophy significant gallbladder dilatation and local, white wall thick- and cell degeneration were present. The length of uninfected ening. Inside the gallbladder of 16 lizards were macroscopi- cells of gallbladder epithelium differed significantly, ranging cally visible masses of small gallstones (about 1–3mmin from 10.30 to 44.20 μm. In the subepithelial connective tis- diameter) and/or debris (Figs. 1a, b). sue, numerous heterophils were observed. All the examined animals were coccidia positive. Oocysts No endogenous stages of the coccidia were detected in were present both in the intestinal contents (11/24) and the bile intestinal samples. Parasitol Res (2016) 115:681–685 683

Fig. 1 Macroscopically visible pathological changes of the gallbladder in infected lizards. Local white wall thickening (a). Gallstones and debris inside the gallbladder (b)

Discussion for the classification of the coccidia is excystation structures (Box et al. 1980). In Choleoeimeria, the sporocyst wall is Traditional taxonomy of coccidian parasites invading reptiles composed of two valves joined by a longitudinal suture (Jirků placed all species together with oocysts that have four dizoic et al. 2002;Paperna2007). sporocysts into the genus Eimeria (Jirků et al. 2009; Levine Based on the results of morphological analysis of mature 1985). This classification was based primarily on the mor- oocysts and the location of the endogenous stages, we con- phology of the exogenous stages (oocysts, sporocysts, sporo- clude that the invasion observed in the present study was zoites), while less focused on information about the life cycles caused by the genus Choleoeimeria. According to literature, and biology of these coccidia. Therefore, recent studies on all species placed within Choleoeimeria are homoxenous and endogenous development, sporocyst excystation structures strictly host specific (Jirků et al. 2009). Therefore, we com- as well as molecular analysis all lead to a reclassification of pared our results only with Eimeria-like coccidia parasitizing some reptilian coccidia (Paperna 2007). In fact, all tetrasporic the Australian agamid from the genus Pogona.Uptonow,no dizoic coccidia parasitizing reptilian hosts have been split into species of Choleoeimeria have been reported in any of the three genera: Eimeria, Acroeimeria and Choleoeimeria (Ten- eight species that belong to the genus Pogona (Cannon ter et al. 2002; Lainson and Paperna 1999). 1967; McAllister et al. 1995;Walden2009). Only one Members of Choleoeimeria have a specific location re- tetrasporocystic dizoic coccidian had been previously de- stricted to the gallbladder epithelium of reptiles, mainly liz- scribed in P. vitticeps. Based solely on the morphology of ards, and less commonly to snakes and tortoises. Usually, the the exogenous stages, Walden (2009) placed them in the ge- epicytoplasmatic development of the endogenous stages leads nus Eimeria and classified as E. pogonae, despite the fact that to hypertrophy of gallbladder epithelium and displacement of the oocysts described by him had features typical of genus host cells into the bile lumen. Mature oocysts contain four Choleoeimeria. Although Walden (2009) suspected that the sporocysts each witch two sporozoites. Oocysts are character- location of the endogenous stages for E. pogonae was in the ized by a lack of micropyle, oocyst residuum, and are gener- epithelium of the gallbladder, he did not determine the site of ally uniform with a typical cylindroidal or ellipsoidal shape infection. Unfortunately, Walden’s Ph.D. thesis was not pub- (length/width ratio 1.6–2.2). Sporocysts have neither the lished in scientific revived journal, but his results should not Stieda nor substieda bodies (Jirků et al. 2009; Sloboda and be ignored; however, taxonomic revision of E. pogonae is Modrý 2006; Tenter et al. 2002). Another important feature needed. In fact, the oocysts of E. pogonae are

Fig. 2 Photographs of ooccyst of Choleoeimaria pogonae. a Sporulated oocyst containing four sporocysts each with two sporozoites. Note compact sporocyst residuum (csr). b Sporulated oocyst isolated from the gallbladder with eight sporozoites released from sporocysts. Note polar granule (pg), posterior refractile body of the sporozoite (prb)and sporozoite nucleus (n). c Line drawings of sporulated oocysts. All in the same scale bar=10 μm 684 Parasitol Res (2016) 115:681–685

hypertrophy of epithelial cells and tissue debris (Sloboda and Modrý 2006). In the present study, only single-epithelial cells in gallbladder were infected with endogenous stages of coccidian, but most of them were hypertrophied. There is limited information on the pathology of reptilian gallbladder; however, the pathological hypertrophy of the mucosa of bil- iary vesicle is frequently identified as the characteristic re- sponse for the presence of gallstones in mammalian (Mathur et al. 2012). It is possible that in dissected animals, most of the epithelial abnormalities in gallbladders were caused by chronic inflammation induced by conglomerates of oocysts accompanied with precipitates of tissue debris. Our results of the histopathological examination confirmed this thesis. The largest hypertrophy of the gallbladder’s mucosa was observed Fig. 3 Various endogenous stages of coccidia. Note infected cells are in 16 lizards with macroscopically visible gallstones or displaced into the lumen of bile duct debris. All these factors may lead to the lack of oocysts in the faecal samples in late patent period and some difficulties in indistinguishable in morphology from those found by the diagnosis of reptile choleoeimeriosis using standard present study, which indicate that these two coccidians belong coprological methods. Our observations indicate low sen- to the same genus—Choleoeimeria. Due to a generally narrow sitivity of faecal flotation. Indeed, in 13 of 24 dissected host specificity of Choleoeimeria spp., the chances that the animals, we observed numerous oocysts only in their gall- coccidian in the present study is different from that of Walden bladders, while results of parasitological investigations of study are extremely low. Therefore, most probably, both faeces or intestinal contents were negative. A similar pro- coccidians are conspecific. Considering morphological and portion of false-negative results was determined by developmental traits, we propose reclassification of Sloboda and Modrý (2006) in a faecal examination of E. pogonae and transfer it into the correct genus as Chamaleo calyptratus. The consequence of such false- Choleoeimeria pogonae comb. nov. negative results of faecal examination is probably an un- Small differences in the size of oocysts and sporocysts in derestimation of the prevalence of Choleoeimeria spp. in- both studies might be related to use of different microscopic vasions in captive and wild reptiles. optic or can be accounted for duration of infection. Duszynski On the other hand, using coprological diagnostic (1971) found that oocysts appearing the earliest in infection methods for detection of Choleoeimeria sp. in early were smaller than those observed later during patency, where- stage of infection can be also problematic, due to rela- as the shape index remained more or less constant. In the tively long prepatent period. As estimated by Sloboda present study, all of the examined bearded dragons were in and Modrý (2006) in their experimental trials, oocysts late stages of infection, which was confirmed by the results of of Choleoeimeria hirbayah were evacuated with the fae- the autopsy and presence of sporulated oocysts in the gallblad- ces within 50–83 days of infection. Several authors also der. In general, sporulation of oocysts in the genus suggest that in early stages of patent period (when oo- Choleoeimeria is endogenous. However, according to cysts are present in faeces) animals usually demonstrate Sloboda and Modrý (2006), localization of this process is time good condition and nutrition status (Abdel-Baki 2014; dependent and may occur in intestine or gallbladder as well as Al-Quraishy et al. 2013; Lainson and Paperna 1999). outside the host. In the initial phase of the patent period, all Because routine parasitological examinations of asymp- oocysts observed in faeces are unsporulated, but the percent- tomatic reptiles are not a common veterinary practice, age of fully sporulated oocysts increases during patency. In most animals are simply not diagnosed. This fact may later stages of infection, most oocysts probably remain in the explain why in P. vitticeps—one of the most common gallbladder, having enough time to sporulate. In our study, the lizards in individual breeding—invasion of the genus majority of oocysts recovered from the gallbladder were fully Choleoeimeria has not been described so far. In contrast sporulated and only 2 % remained unsporulated, while in to invasion of I. amphiboluri, which is usually associ- Walden’s study, 20 % of oocysts isolated from fresh faeces ated with clinical signs such as diarrhea and progressive samples were unsporulated, which suggests an early stage of apathy in captive bearded dragons (Raiti 2012). The infection. pathogenicity potential of C. pogonae is not clear for In heavily infected animals, oocyst shedding can be totally us. To establish accurate disease state associated with stopped by obturation of the bile ducts caused by pathological biliary coccidiosis in bearded dragons of different ages Parasitol Res (2016) 115:681–685 685 and different stages of invasion, clinical and pathologi- Jirků M, Jirků M, Oborník M, Lukes J, Modrý D (2009) A model for cal data conducted in experimental trials are necessary. taxonomic work on homoxenous coccidia: redescription, host spec- ificity, and molecular phylogeny of Eimeria ranae Dobell, 1909, with a review of anuran-host Eimeria (Apicomplexa: ). J Euk Microbiol 56:39–51 Open Access This article is distributed under the terms of the Creative Lainson R, Paperna I (1999) Some coccidia from the gall bladder Commons Attribution 4.0 International License (http:// and intestine of the teiid lizard Ameiva ameiva ameiva and creativecommons.org/licenses/by/4.0/), which permits unrestricted use, the gecko Hemidactylus mabouia in North Brazil. Parasite 6: distribution, and reproduction in any medium, provided you give appro- 151–162 priate credit to the original author(s) and the source, provide a link to the Levine ND (1985) The phylum Apicomplexa. CRC Press, Boca Raton, Creative Commons license, and indicate if changes were made. Florida MathurSK,DuhanA,SinghS,AggarwalM,AggarwalG,Sen R, Singh S, Garg S (2012) Correlation of gallstone charac- References teristics with mucosal changes in gall bladder. Trop Gastroenterol 33:39–44 McAllister CT, Upton SJ, Jacobson ER, Kopit W (1995) A description of Abdel-Baki AS (2014) Description of Choleoeimeria duszynskii n. sp. Isospora amphiboluri (Apicomplexa: Eimeriidae)fromtheinland (Apicomplexa: Eimeriidae) from the gall bladder of the Middle bearded dragon, Pogona vitticeps (Sauria: Agamidae). J Parasitol Eastern short-fingered gecko Stenodactylus doriae (Blanford) 81:281–284 (Sauria: Gekkonidae) in Saudi Arabia. Syst Parasitol 87:299–304 Paperna I (2007) Ultrastructural review of Choleoeimeria spp., a coccid- Abdel-Baki AS, Al-Quraishy S, Duszynski DW (2014) Choleoeimeria ium infecting the gall-bladder epithelium of reptiles. Parassitologia salaselensis sp. n. (Apicomplexa: Eimeriidae) from the gall bladder 49:247–256 of the horned viper Cerastes gasperettii (Serpentes: Viperidae)in Saudi Arabia. Folia Parasitol 61:201–205 Paperna I, Landsberg JH (1989) Description and taxonomic discussion of Al-Quraishy S, Abdel-Baki AS, Al Otaibi MSA (2013) Choleoeimeria eimerian coccidia from African and Levantine geckoes. S Afr J Zool – bunopusi sp. n. (Apicomplexa: Eimeriidae) infecting the gall bladder 24:345 355 of the tuberculated gecko Bunopus tuberculatus (Reptilia: Raiti P (2012) Husbandry, diseases, and veterinary care of the bearded – Gekkonidae) from Saudi Arabia. Acta Protozool 52:267–272 dragon (Pogona vitticeps). J Herp Med and Surg 22:117 131 Box ED, Marchiondo AA, Duszynski DW, Davis CP (1980) Ultrastructure of Sloboda M, Modrý D (2006) New species of Choleoeimeria sporocysts from passerine birds and opossums: comments (Apicomplexa: Eimeriidae) from the veiled , on classification of the genus Isospora. J Parasitol 66:68–74 Chamaeleo calyptratus (Sauria: Chamaeleonidae), with taxo- Cannon LRG (1967) New coccidia from Australian lizards. I. Isospora. nomic revision of eimerian coccidia from . Folia Parasitology 57:227–235 Parasitol 53:91–97 Duszynski DW (1971) Increase in size of Eimeria separata oocysts dur- Tenter AM, Barta JR, Beveridge I, Duszynski DW, Mehlhorn H, ing patency. J Parasitol 57:948–952 Morrison DA, Thompson RC, Conrad PA (2002) The conceptual Duszynski DW, Wilber PG (1997) Guidelines for publishing new species basis for a new classification of the coccidia. Int J Parasitol 32:595– descriptions of eimerians (Apicomplexa: Eimeriidae). J Parasitol 83: 616 333–336 Walden MR (2009) Characterizing the epidemiology of Isospora Jirků M, Modrý D, Slapeta JR, Koudela B, Lukes J (2002) The phylogeny amphiboluri in captive bearded dragons (Pogona vitticeps). Ph. D. of Goussia and Choleoeimeria (Apicomplexa: Eimeriorina) and the thesis, Louisiana State University, Baton Rouge, Louisiana, USA evolution of excystation structures in coccidia. Protist 153:379–390 etd-05292009-214931